Some recent data on sperm morphology and motility kinetics in Atlantic cod (Gadus morhua L.).

Studying biology of sperm provides valuable information to optimize artificial reproduction and is crucial for sustainable aquaculture. Here, we investigated morphology of spermatozoon in Atlantic cod (Gadus morhua) using transmission and scanning electron microscopy. Furthermore, spermatozoa motility kinetics at different osmolalities were studied using computer-assisted sperm analysis software. The spermatozoon lacked an acrosome and consisted of a head, midpiece, and flagellum. The head of spermatozoa was round, oval, and rather elongated in shape, showing high variations in dimensions. There were up to 6 mitochondria that encircled the proximal part of the flagellum. The proximal and distal centrioles were located within the nuclear notch and arranged orthogonal to each other. The axoneme had a typical 9 + 2 microtubule structure. The flagellar length of spermatozoon was 66.94 ± 0.46 µm. Spermatozoa were immotile in the seminal plasma. Dilution of sperm with natural seawater (1100 mOsmol/kg) resulted in initiation of motility for 91.0 ± 3.4% of spermatozoa with average velocity of 86.2 ± 2.3 µm/s and beating frequency of 52 Hz. The duration of spermatozoa motility was > 6 min; however, the percentage of motile spermatozoa decreased at 60 s post-activation. When osmolality of natural seawater was modified using distilled water or NaCl, spermatozoa motility was not initiated at ≤ 400 and ≥ 2500 mOsmol/kg, and the highest percentage of motility was observed at 730-1580 mOsmol/kg. In a sucrose solution, spermatozoa motility was initiated and suppressed at 600 and 1500 mOsmol/kg, respectively, and highest percentage of motility was observed at 800-1100 mOsmol/kg. Spermatozoon morphology comparisons within Gadiformes showed differences in dimensions of head and mitochondria, flagellar length, and number of mitochondria. The present study provides valuable data that can be used for phylogenetic implications based on spermatozoon morphology and for development of artificial fertilization and sperm cryopreservation protocols based on sperm motility.

Nesfatin-1-like peptide suppresses hypothalamo-pituitary-gonadal mRNAs, gonadal steroidogenesis, and oocyte maturation in fish†.

Nucleobindin (Nucb)-1 and Nucb2 are DNA and Ca2+ binding proteins with multiple functions in vertebrates. Prohormone convertase-mediated processing of Nucb2 results in the production of biologically active nesfatin-1. Nesfatin-1 is involved in the regulation of reproduction in many vertebrates, including fish. Our lab originally reported a nesfatin-1-like peptide (Nlp) encoded in Nucb1 that exhibits nesfatin-1-like metabolic effects. We hypothesized that Nlp has a suppressive role in the reproductive physiology of fish. In this research, whether Nlp regulates reproductive hormones and oocyte maturation in fish were determined. Single intraperitoneal (IP) injection of goldfish Nlp (50 ng/g body weight) suppressed salmon and chicken gonadotropin-releasing hormone (sgnrh and cgnrh2), gonadotropin-inhibiting hormone (gnih) and its receptor (gnihr), and kisspeptin and brain aromatase mRNA expression in the hypothalamus of both male and female goldfish. In the pituitary, Nlp decreased mRNAs encoding lhb, fshb and kisspeptin and its receptor, while a significant increase in gnih and gnihr was observed. In the gonads, lh (only in male fish) and fsh receptor mRNAs were also significantly downregulated in Nlp-injected fish. Sex-specific modulation of gnih, gnihr, and kisspeptin system in the gonads was also observed. Nlp decreased sex steroidogenic enzyme encoding mRNAs and circulating levels of testosterone and estradiol. In addition, incubation of zebrafish ovarian follicles with Nlp resulted in a reduction in oocyte maturation. These results provide evidence for a robust role for Nlp in regulating reproductive hormones in goldfish and oocyte maturation in zebrafish, and these effects resemble that of nesfatin-1.

Metabolic stress leads to divergent changes in the ghrelinergic system in goldfish (Carassius auratus) gonads.

Various endocrine factors that regulate energy homeostasis are also implicated in the reproductive physiology of mammals. However, the hormonal link between metabolism and reproduction in fish is poorly understood. Ghrelin is a multifunctional hormone with both metabolic and reproductive roles in vertebrates. Post-translational acylation by ghrelin-O-acyltransferase (GOAT) is critical for its biological actions. The expression of ghrelin, ghrelin or growth hormone secretagogue receptor (GHSR), and GOAT (which forms the ghrelinergic system) in fish under metabolic stress remains unclear. In this research, we used RT-qPCR and Western blot analysis to determine the expression of the ghrelinergic system in goldfish (during the reproductively active phase) hypothalamus and gonads under 7 and 28 days of fasting. We found a significant increase in preproghrelin mRNA expresson in the ovary, and GOAT mRNA expression in the testis of goldfish deprived of food for 7 days. In fish deprived of food for 28 days, preproghrelin, GHSR and GOAT mRNA expression was significantly increased in the hypothalamus of male goldfish. Such differences were not observed in the hypothalamus of female fish, and in the testis of 28 days fasted fish. Meanwhile, preproghrelin, GHSR, and GOAT expression (both mRNA and protein) was significantly increased in the ovary of female fish fasted for 28 days. Ghrelin has been shown to suppress oocyte maturation in fish. The upregulation of a system that has ovarian inbititory roles suggests a role for ghrelin in maintaining reduced reproductive capability during metabolically challenging periods.

Influence of Probiotics Administration on Gut Microbiota Core: A Review on the Effects on Appetite Control, Glucose, and Lipid Metabolism.

An increasing number of studies has shown that dietary probiotics exert beneficial health effects in both humans and animals. It is well established that gut microbiota play a pivotal role in regulating host metabolism, and a growing number of studies has elucidated that probiotics positively interfere with gut microbiota. Accumulating evidence shows that probiotics, through their metabolic activity, produce metabolites that in turn contribute to positively affect host physiology. For these reasons, probiotics have shown significant potential as a therapeutic tool for a diversity of diseases, but the mechanisms through which probiotics act has not been fully elucidated yet. The goal of this review was to provide evidence on the effects of probiotics on gut microbiota changes associated with host metabolic variations, specifically focusing on feed intake and lipid and glucose metabolism. In addition, we review probiotic interaction with the gut microbiota. The information collected here will give further insight into the effects of probiotics on the gut microbiota and their action on metabolite release, energy metabolism, and appetite. This information will help to improve knowledge to find better probiotic therapeutic strategies for obesity and eating disorders.

Ultrasonography reveals in vivo dose-dependent inhibition of end systolic and diastolic volumes, heart rate and cardiac output by nesfatin-1 in zebrafish.

Nesfatin-1 is an 82 amino acid peptide that inhibits food intake in rodents and fish. While endogenous nesfatin-1, and its role in the regulation of food intake and hormone secretion has been reported in fish, information on cardiovascular functions of nesfatin-1 in fish is in its infancy. We hypothesized that cardiac NUCB2 expression is meal responsive and nesfatin-1 is a cardioregulatory peptide in zebrafish. NUCB2/nesfatin-1 like immunoreactivity was detected in zebrafish cardiomyocytes. Real-time quantitative PCR analysis found that the cardiac expression of NUCB2A mRNA in unfed fish decreased at 1h post-regular feeding time. Food deprivation for 7days did not change NUCB2A mRNA expression. However, NUCB2B mRNA expression was increased in the heart of zebrafish after a 7-day food deprivation. Ultrasonography of zebrafish heart at 15min post-intraperitoneal injection of nesfatin-1 (250 and 500ng/g body weight) showed a dose-dependent inhibition of end diastolic and end systolic volumes. A dose dependent decrease in heart rate and cardiac output was observed in zebrafish that received nesfatin-1, but no changes in stroke volume were found. Nesfatin-1 treatment caused a significant increase in the expression of Atp2a2a mRNA encoding the calcium-handling pump, SERCA2a, while it had no effects on the expression of calcium handling protein RyR1b encoding mRNA. Our data support cardiosuppressive effects of nesfatin-1 in zebrafish, and reveals energy availability as one determinant of cardiac NUCB2 mRNA expression.

Nutrient status modulates the expression of nesfatin-1 encoding nucleobindin 2A and 2B mRNAs in zebrafish gut, liver and brain.

Nesfatin-1 is a naturally occurring, 82-amino acid peptide processed from the precursor nucleobindin 2 (NUCB2), a highly conserved protein among vertebrates. In fish, two isoforms of NUCB2 (NUCB2A and NUCB2B) exist, and nesfatin-1 has been identified in goldfish and Ya fish. We recently reported the presence and appetite suppressing effects of nesfatin-1 in goldfish. The main objectives of this study were to characterize NUCB2 in zebrafish, and determine whether NUCB2 mRNAs are affected by food availability. Tissue distribution of NUCB2A and NUCB2B mRNAs, and NUCB2/nesfatin-1-like immunoreactivity (ir) in the gut of zebrafish were also investigated. In zebrafish, nesfatin-1 region (1-82 amino acids) in NUCB2A is 78% identical to NUCB2B. Both NUCB2A and NUCB2 mRNAs were most abundant in the liver, while less expression was found in other tissues including the brain and gut. NUCB2/nesfatin-1-like immunoreactivity was detected in the mucosal layer cells of zebrafish anterior gastrointestinal tract. NUCB2A and NUCB2B mRNA expression were decreased in the brain of zebrafish 3h after feeding, and after a 7-day food deprivation. Both NUCB2A and NUCB2B mRNAs in the gut were also decreased following 7 days of food deprivation, while NUCB2B mRNA was increased in the liver. Our results provide molecular and functional evidences to support potential anorectic and metabolic roles for endogenous nesfatin-1 in zebrafish. To our knowledge, this is the first report on NUCB2B characterization in vertebrates.

Protease in sturgeon sperm and the effects of protease inhibitors on sperm motility and velocity.

In mammals, proteases are present in sperm acrosome and play key role in fertilization. Sturgeon sperm has an acrosome, but its physiology, biochemistry, and potential role in fertilization are unknown. In the present study, we have observed high protease activity in acidic extract of intact sperm compared to that of seminal plasma in sterlet (Acipenser ruthenus). The protease activity was decreased and increased in acidic extract of motility-activated sperm and in the activation medium, respectively. Molecular analysis revealed total protease and serine (acrosin) protease activities in sperm acidic extract which was accumulated in a protein band with relative molecular mass of 35 kDa. Immunoelectron microscopy using an affinity-purified polyclonal antibody for boar acrosin localized the protease at the acrosome region. Moreover, initiation of sperm motility was inhibited after activation in the presence of inhibitors for both trypsin-like and chymotrypsin-like proteases, while the effects of protease inhibitors on sperm velocity were uncertain. Our results indicate similarities in physiology and biochemistry of acrosome between sturgeon and mammals and suggest potential role of protease in the initiation of sperm motility in sturgeon.

Impact of absolute food deprivation on the reproductive system in male goldfish exposed to sex steroids.

There is a link between metabolism and reproduction as metabolic hormones affect hypothalamus-pituitary-testis (HPT) hormonal functions and vice versa. The aim of the present study was to investigate the effects of negative energy balance on the reproductive system in male goldfish exposed to testosterone (T) and 17ß-estradiol (E2). Following 7 days of food deprivation (FD), ANOVA models showed significant FD × sex steroid interactions on sperm quality and circulating sex steroid levels. When FD effects were investigated, 11-ketotestosterone (11-KT) level and sperm motility and velocity decreased in food-deprived goldfish in the control group. In E2-exposed goldfish, FD decreased sperm production in addition to sperm motility and velocity that coincided with an elevation of circulating E2 level. However, FD did not significantly impact sex steroids and sperm quality in T-exposed goldfish. ANOVA models showed non-significant FD × sex steroid interactions for HSI, GSI, circulating luteinizing hormone (Lh) level, and metabolic (preproghrelin, goat and nucb2) and reproductive (kiss1, gpr54 and gnrh3) mRNAs. Furthermore, results showed that FD decreased HSI, and increased Lh levels and testicular preproghrelin and goat mRNAs, while sex steroids increased mid-brain nucb2, kiss1 and gpr54 mRNAs. Together, our results suggest that FD-induced inhibition of androgenesis resulted in diminished sperm quality associated with activation of the testicular ghrelinergic system, and negative feedback of 11-KT increased Lh level. The FD-induced testicular metabolic and hormonal system was impacted in goldfish exposed to sex steroids. However, the negative effects of FD on sperm quality were accelerated in E2-exposed goldfish due to estrogenic activity. This study provides novel information to better understand metabolic-associated reproductive disorders in fish.

Adverse effects of bisphenol A on reproductive physiology in male goldfish at environmentally relevant concentrations.

Alternations of reproductive physiology were studied in the male goldfish (Carassius auratus L.) exposed to environmentally relevant concentrations (0.6, 4.5 and 11.0 µg/L) of bisphenol A (BPA) at days 10, 20 and 30 after exposure. Significant effects of BPA concentration, exposure time and their interactions were observed on testosterone (T), 11-ketotestosterone (11-KT) and sperm motility and velocity, but gonadosomatic index (GSI), hepatosomatic index (HSI) and 17ß-estradiol (E(2)) were not affected. Vitellogenin (VTG) was only affected by BPA concentration. The T and 11-KT levels were significantly decreased in the BPA-treated groups after 20 or 30 days. Sperm motility was significantly decreased at 15, 30, 60 and 90 s post-activation in the BPA-treated groups after 20 or 30 days. But, significant decrease in sperm velocity was observed at 30, 60 and 90 s post-activation in the BPA-treated groups at all exposure times. The VTG was significantly increased in the males exposed to 11.0 µg/L at day 30 after exposure. The GSI, HSI and E(2) did not differ between the BPA-treated groups and control. The present study shows that the decrease of sperm quality is concurrent with the decrease of androgens and increase of VTG. The results suggest adverse effects of BPA on sperm motility and velocity via modifications of testicular steroidogenesis that might correspond to alternation in sperm maturation.

Sperm characteristics and androgens in Acipenser ruthenus after induction of spermiation by carp pituitary extract or GnRHa implants.

Spermiation and changes in androgen (testosterone, T and 11-ketotestosterone, 11-KT) levels were studied in sterlet (Acipenser ruthenus) treated with GnRH agonist implants (DAla(6)-Pro(9)-LHRHa) at 25 and 75 µg kg(-1) b.w. and compared with those males treated with 4 mg kg(-1) b.w. of carp pituitary extract (CPE) and 3 pellets of Ovopel kg(-1) b.w., which contains DAla(6)-Pro(9)NEt-mGnRH and metoclopramide. Sperm quality (sperm mass, spermatozoa concentration and sperm motility and velocity) was evaluated 24, 48 and 72 h after hormonal treatments. Males did not release sperm in the control group injected with physiological solution, while sperm could not be collected 7 days after treatments in all hormonally treated groups. Spermiation rates were 100 % in the CPE and Ovopel groups and 25-50 % in the GnRHa-treated groups. Sperm production was significantly lower in the GnRHa-treated groups than in the CPE and Ovopel groups and decreased 72 h after hormonal treatment. Sperm motility and velocity were higher in the Ovopel and GnRHa (75 µg) groups compared to the CPE and GnRHa (25 µg) groups and decreased 72 h after hormonal treatment. Androgens were only affected in spermiating males and changed in the Ovopel and GnRHa (75 µg) after hormonal treatment. Significant correlations were observed between sperm production, sperm motility and sperm velocity, but not androgens. The present study suggests involvement of dopamine in sturgeon spawning. Additionally, better sperm quality observed in the Ovopel group and particularly sperm motility in the GnRHa (75 µg) suggests enhancement of sperm quality in sturgeon treated with GnRHa. Therefore, further study is needed to induce fully spermiation using GnRHa implants in combination with a dopamine inhibitor.

A chronic exposure to bisphenol A reduces sperm quality in goldfish associated with increases in kiss2, gpr54, and gnrh3 mRNA and circulatory LH levels at environmentally relevant concentrations.

The bisphenol A (BPA)-disrupted reproductive functions have been demonstrated in male animals. In fish, it has been shown that environmentally relevant concentrations of BPA decrease sperm quality associated with inhibition of androgen biosynthesis. However, BPA effects on neuroendocrine regulation of reproduction to affect testicular functions are largely unknown. In the present study, reproductive functions of hypothalamus and pituitary were studied in mature male goldfish exposed to nominal 0.2, 2.0 and 20.0 µg/L BPA. At 90 d of exposure, sperm volume, velocity, and density and motility were decreased in goldfish exposed to 0.2, 2.0, and 20.0 µg/L BPA, respectively (p < 0.05). At 30 d of exposure, there were no significant changes in circulatory LH levels and mRNA transcripts of kiss1, Kiss2, gpr54, and gnrh3. At 90 d of exposure, circulatory LH levels showed trends toward increases in BPA exposed goldfish, which was significant in those exposed to 2.0 µg/L (P < 0.05). At this time, Kiss2, gpr54, and gnrh3 mRNA levels were increased in goldfish exposed to any concentrations of BPA (p < 0.05). This study shows that BPA-diminished sperm quality was accompanied by an increase in circulatory LH levels associated with increases in mRNA transcripts of upstream neuroendocrine regulators of reproduction in goldfish. Further, this is the first study to report circulatory levels of LH in fish exposed to BPA.

A Review on Environmental Contaminants-Related Fertility Threat in Male Fishes: Effects and Possible Mechanisms of Action Learned from Wildlife and Laboratory Studies.

Increasing global rates of diminished fertility in males has been suggested to be associated with exposure to environmental contaminants (ECs). The aquatic environments are the final repository of ECs. As the reproductive system is conserved in vertebrates, studies on the effects of ECs on fertility endpoints in fishes provide us with valuable information to establish biomarkers in risk assessment of ECs, and to understand the ECs-related fertility threat. The aim of the present review was to evaluate associations between ECs and fertility determinants to better understand ECs-related male fertility threat in male fishes. Wildlife studies show that the reproductive system has been affected in fishes sampled from the polluted aquatic environment. The laboratory studies show the potency of ECs including natural and synthetic hormones, alkylphenols, bisphenols, plasticizers, pesticides, pharmaceutical, alkylating, and organotin agents to affect fertility determinants, resulting in diminished fertility at environmentally relevant concentrations. Both wildlife and laboratory studies reveal that ECs adverse effects on male fertility are associated with a decrease in sperm production, damage to sperm morphology, alternations in sperm genome, and decrease in sperm motility kinetics. The efficiency of ECs to affect sperm quality and male fertility highly depends on the concentration of the contaminants and the duration of exposure. Our review highlights that the number of contaminants examined over fertility tests are much lower than the number of contaminants detected in our environment. The ECs effects on fertility are largely unknown when fishes are exposed to the contaminants at early developmental stages. The review suggests the urgent need to examine ECs effects on male fertility when a fish is exposed at different developmental stages in a single or combination protocol. The ECs effects on the sperm genome are largely unknown to understand ECs-related inheritance of reproductive disorders transmitted to the progeny. To elucidate modes of action of ECs on sperm motility, it is needed to study functional morphology of the motility apparatus and to investigate ECs-disrupted motility signaling.

Nesfatin-1 is an inhibitor of the growth hormone-insulin-like growth factor axis in goldfish (Carassius auratus).

Nesfatin-1, an 82 amino acid peptide cleaved from the N-terminal of its precursor nucleobindin-2 (NUCB2), is emerging as a multifunctional peptide in fish. The present study aimed to determine whether nesfatin-1 plays a role in fish somatic growth by modulating the growth hormone (GH)/insulin-like growth factor (IGF) axis, using a representative teleost model, the goldfish (Carassius auratus). The results demonstrated that a single i.p. injection of synthetic goldfish nesfatin-1 significantly decreased the expression of hypothalamic pacap (approximately 90%) and pituitary Gh (approximately 90%) mRNAs at 15 minutes post-injection. Serum GH levels were also reduced as a result of nesfatin-1 administration, by approximately 45% and 55% at 15 and 30 minutes post-injection, respectively. Likewise, in vitro treatment of goldfish dispersed pituitary cells with nesfatin-1 reduced Gh secretion, suggesting that nesfatin-1 acts directly on pituitary somatotrophs to inhibit Gh release. Exposure of cultured liver fragments to nesfatin-1 (0.1, 1 and 10 nmol L-1 ) led to a significant reduction in igf-1 mRNA at 120 minutes and of igf-II mRNA at 30 and 60 minutes post-incubation. Collectively, these results indicate a suppressive role for nesfatin-1 on the goldfish GH/IGF axis. Immunohistochemical studies demonstrated that NUCB2/nesfatin-1-like immunoreactivity, although present in the goldfish pituitary, is not colocalised with GH in goldfish somatotrophs. Thus, nesfatin-1 does not appear to act in an autocrine manner to regulate GH secretion. Taken together, this research found that the pituitary gland is an important source of endogenous NUCB2/nesfatin-1 and also that nesfatin-1 directly suppresses the Gh/IGF axis in goldfish.

Feeding and food availability modulate brain-derived neurotrophic factor, an orexigen with metabolic roles in zebrafish.

Emerging findings point to a role for brain-derived neurotrophic factor (BDNF) on feeding in mammals. However, its role on energy balance is unclear. Moreover, whether BDNF regulates energy homeostasis in non-mammals remain unknown. This research aimed to determine whether BDNF is a metabolic peptide in zebrafish. Our results demonstrate that BDNF mRNAs and protein, as well as mRNAs encoding its receptors trkb2, p75ntra and p75ntrb, are detectable in the zebrafish brain, foregut and liver. Intraperitoneal injection of BDNF increased food intake at 1, 2 and 6 h post-administration, and caused an upregulation of brain npy, agrp and orexin, foregut ghrelin, and hepatic leptin mRNAs, and a reduction in brain nucb2. Fasting for 7 days increased bdnf and p75ntrb mRNAs in the foregut, while decreased bdnf, trkb2, p75ntra and p75ntrb mRNAs in the brain and liver. Additionally, the expression of bdnf and its receptors increased preprandially, and decreased after a meal in the foregut and liver. Finally, we observed BDNF-induced changes in the expression and/or activity of enzymes involved in glucose and lipid metabolism in the liver. Overall, present results indicate that BDNF is a novel regulator of appetite and metabolism in fish, which is modulated by energy intake and food availability.

Composition of ovarian fluid in endangered Caspian brown trout, Salmo trutta caspius, and its effects on spermatozoa motility and fertilizing ability compared to freshwater and a saline medium.

In this study, ovarian fluid composition and its effects on the motility and fertilizing ability of sperm were studied in endangered Caspian brown trout, Salmo trutta caspius, and were compared with a saline activation medium (125 mM NaCl, 30 mM Glycine, 20 mM Tris-HCl, pH = 9.0) and freshwater as the control. The ovarian fluid was composed of sodium 164.4 +/- 4.4 mM l(-1), potassium 1.8 +/- 0.1 mM l(-1), calcium 0.6 +/- 0.1 mM l(-1), magnesium 0.4 +/- 0.02 mM l(-1), chloride 127.4 +/- 5.9 mM l(-1), total protein 389.5 +/- 89.6 mg 100 ml(-1), cholesterol 9.3 +/- 1.2 mg dl(-1), and glucose 3.3 +/- 0.2 mM l(-1). The percentage of motile spermatozoa and the duration of sperm motility were significantly higher in ovarian fluid (62 +/- 3%, 74.6 +/- 0.8 s) than freshwater (35 +/- 4%, 44 +/- 1 s), but they did not differ significantly from saline medium (56 +/- 3%, 74.3 +/- 0.7 s) (P > 0.05). Higher eyeing rates were observed after the activation of sperm in ovarian fluid and saline solution than freshwater when 35,000 or 350,000 spermatozoa per egg were added into the activation media. However, no significant differences were observed at higher concentrations of spermatozoa per egg (730,000) (P > 0.05). Also, this study showed that the ovarian fluid composition can be considered as a species-specific character among salmonid fishes. As a conclusion, the results of this study recommend the use of ovarian fluid or the saline solution as an activation medium in the artificial reproduction of Caspian brown trout.

Metabolic hormones and the regulation of spermatogenesis in fishes.

Metabolic hormones play essential regulatory roles in many biological processes, including morphogenesis, growth, and reproduction through the maintenance of energy balance. Various metabolic hormones originally discovered in mammals, including ghrelin, leptin, and nesfatin-1 have been identified and characterized in fish. However, physiological roles of these metabolic hormones in regulating reproduction are largely unknown in fishes, especially in males. While the information available is restricted, this review attempts to summarize the main findings on the roles of metabolic peptides on the reproductive system in male fishes with an emphasis on testicular development and spermatogenesis. Specifically, the primary goal is to review the physiological interactions between hormones that regulate reproduction and hormones that regulate metabolism as a critical determinant of testicular function. A brief introduction to the localization of metabolic hormones in fish testis is also provided. Besides, the consequences of fasting and food deprivation on testicular development and sperm quality will be discussed with a focus on interactions between metabolic and reproductive hormones.

Dietary lipid content reorganizes gut microbiota and probiotic L. rhamnosus attenuates obesity and enhances catabolic hormonal milieu in zebrafish.

In the present study, we explored whether dietary lipid content influences the gut microbiome in adult zebrafish. Diets containing three different lipid levels (high [HFD], medium [MFD], and low [LFD]) were administered with or without the supplementation of Lactobacillus rhamnosus (P) to zebrafish in order to explore how the dietary lipid content may influence the gut microbiome. Dietary lipid content shifted the gut microbiome structure. The addition of L. rhamnosus in the diets, induced transcriptional reduction of orexigenic genes, upregulation of anorexigenic genes, and transcriptional decrease of genes involved in cholesterol and triglyceride (TAG) metabolism, concomitantly with lower content of cholesterol and TAG. Probiotic feeding also decreased nesfatin-1 peptide in HFD-P and attenuated weight gain in HFD-P and MFD-P fed zebrafish, but not in LFD-P group. Intestinal ultrastructure was not affected by dietary fat level or probiotic inclusion. In conclusion, these findings underline the role of fat content in the diet in altering gut microbiota community by shifting phylotype composition and highlight the potential of probiotics to attenuate high-fat diet-related metabolic disorder.

In vivo efficacy, toxicity and biodistribution of ultra-long circulating desferrioxamine based polymeric iron chelator.

Desferrioxamine (DFO) is currently in clinical use to remove iron from transfusion-dependent patients with ß-thalassemia major, sickle-cell anemia and the myelodysplastic syndromes. However, its short half-life, burdensome, subcutaneous mode of administration and propensity to cause neurotoxicity at high doses greatly hinder its use. Thus, developing an optimized version of DFO with extended half-life, and reduced toxicity is a major goal. Using high molecular weight (MW), non-toxic, hyperbranched polyglycerol with high functionality, we demonstrate that the efficacy of DFO can be tuned with considerable reduction in toxicity. Using zebrafish embryos and mice, we tested toxicity, iron removal efficacy with low dosing and the biodistribution of ultra-long circulating DFO (ULC-DFO) conjugates. There was no significant difference in the mortality and development of zebrafish embryos upon exposure to ULC-DFO. Similarly, body weights and serum lactate dehydrogenase levels in mice treated with ULC-DFO remained within the normal range throughout the tolerance study. Moreover, ULC-DFO is significantly more effective than low MW DFO in promoting iron removal both from organs and via urine in iron overloaded mice despite using a moderate, once-weekly dosing schedule. This is probably due to the extended circulation half-life of ULC-DFO. The MW of ULC-DFO influences the accumulation and biodistribution, with highest MW (637 KDa) associated with up to 12% accumulation in the liver. In contrast, ULC-DFO with MWs of 75 KDa and lower were associated with relatively low organ accumulation, indicating that biodistribution of ULC-DFO can be tuned. Since ULC-DFO has improved iron removal properties, longer plasma retention time and possesses excellent biocompatibility, it represents a polymer conjugate with high clinical utility in comparison to DFO for the treatment of transfusion dependent iron overload. More importantly, ULC-DFO is anticipated to reduce the requirement for prolonged subcutaneous infusion of DFO.

Ghrelin O-Acyl Transferase in Zebrafish Is an Evolutionarily Conserved Peptide Upregulated During Calorie Restriction.

Ghrelin is a multifunctional orexigenic hormone with a unique acyl modification enabled by ghrelin O-acyl transferase (GOAT). Ghrelin is well-characterized in nonmammals, and GOAT sequences of several fishes are available in the GenBank. However, endogenous GOAT in non-mammals remains poorly understood. In this research, GOAT sequence comparison, tissue-specific GOAT expression, and its regulation by nutrient status and exogenous ghrelin were studied. It was found that the bioactive core of zebrafish GOAT amino acid sequence share high identity with that of mammals. GOAT mRNA was most abundant in the gut. GOAT-like immunoreactivity (i.r.) was found colocalized with ghrelin in the gastric mucosa. Food deprivation increased, and feeding decreased GOAT and preproghrelin mRNA expression in the brain and gut. GOAT and ghrelin peptides in the gut and brain showed corresponding decrease in food-deprived state. Intraperitoneal injection of acylated fish ghrelin caused a significant decrease in GOAT mRNA expression, suggesting a feedback mechanism regulating its abundance. Together, these results provide the first in-depth characterization of GOAT in a non-mammal. Our results demonstrate that endogenous GOAT expression is responsive to metabolic status and availability of acylated ghrelin, providing further evidences for GOAT in the regulation of feeding in teleosts.

Ovarian structure protein 1: A sensitive molecular biomarker of gonadal intersex in female Japanese medaka after androgen exposure.

Intersex in gonochoristic fish can be induced after exposure to androgens and estrogens. The main objective of the present study was to identify biomarkers that would be predictive of intersex in Japanese medaka (Oryzias latipes) after exposure to synthetic hormones. First a gene was identified, ovarian structure protein 1 (osp1), with strong female-specific expression during gonadal differentiation. The authors hypothesized that osp1 expression would decrease to male levels in females after the exposure of larvae (15-25 d postfertilization [dpf]) to 17ß-trenbolone (TRB; 5 ng/L) and would increase to female levels in males exposed to 17α-ethinylestradiol (EE2; 5 ng/L) and that gonadal intersex would be induced later in life (60 dpf). Tissue distribution and cellular localization of OSP1 was investigated using Western blot and immunohistochemistry. The results indicate that this exposure regime delays testicular maturation in males and development of ovarian intersex in females. Although decreased osp1 expression in females exposed to TRB correlated to changes in ovarian phenotype, up-regulation of osp1 was not observed in males exposed to EE2. In addition, OSP1 was only observed in ovaries and localized in the cytoplasm and follicular layer of immature and mature oocytes. The authors conclude that osp1 is a promising biomarker of androgen exposure and gonadal intersex in female medaka.